Figure 4.

Identification of Plk3 as a CTD Thr4-specific kinase. (A) Immunoprecipitated Pol IIA from HeLa cell extracts was used as substrate for DNA-Pk, Plk3, and Cdk9, and subsequently analysed by western blot with mAbs specific for CTD modifications Thr4-P and Ser2-P, or Rpb1 (POL 3/3 and 1C7, latter recognizes non-modified CTD). (B) Knockdown of Plk3 by siRNA reduces Plk3 protein levels in HeLa cells and concomitantly the levels of Thr4-P in Pol II CTD. (C) Treatment of HeLa cells with 0.2 mM H₂O₂ induces increased Plk3 protein levels after 30 min and concomitantly a significant increase in Thr4-P levels, while Ser2-P, Ser5-P, and Ser7-P levels remained unaffected. Experiments were performed with at least three replicates; error bars show standard deviation.